The objectives of the proposed research are to isolate and characterize the origin of chromosomal replication in the bacterium Escherichia coli and to gain insight into the mechanism of initiation of replication. Restriction nucleases will be used to fragment the E. coli chromosome pulse-labeled at the beginning of a round of replication to obtain defined DNA fragments containing the origin. Amino acid starvation, rifampicin treatment, and temperature shift of temperature sensitive initiation mutants (dnaA and dnaC) will be used to align the chromosomes at the start of replication, both to ascertain the origin fragment and to compare the effects of these diverse treatments on initiation at the molecular level. DNA fragments containing the origin will be cloned for further physical and functional studies, with the goal of determining the base sequence of the origin. Membrane binding of the origin will be examined by using restriction nucleases to cleave and release the portions of the chromosome not specifically bound to membrane in isolated folded chromosomes.